Very large scale immobilized polymer synthesis

• US 5,424,186 A
• Filed: 12/06/1991
• Issued: 06/13/1995
• Est. Priority Date: 06/07/1989
• Status: Expired due to Term

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymax Incorporated
• Inventors: Pirrung, Michael C., Fodor, Stephen P. A., Stryer, Lubert, Read, J. Leighton
• Primary Examiner(s): Kepplinger, Esther M.
• Assistant Examiner(s): Green, Lora M.
• Application Number:
  US07/805,727
• Time in Patent Office: 1,285 Days
• Field of Search: 530/333-337, 436/518, 436/527-532, 436/809, 435/6, 357/4, 427/2, 536/25.3, 536/25.31
• US Class Current: 506/30
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00315 Microtiter plates
• CPC Class Codes: B01J 2219/00432 Photolithographic masks
• CPC Class Codes: B01J 2219/00434 Liquid crystal masks
• CPC Class Codes: B01J 2219/00436 Maskless processes
• CPC Class Codes: B01J 2219/00459 Beads
• CPC Class Codes: B01J 2219/00468 by manipulation of individual beads
• CPC Class Codes: B01J 2219/00475 Sheets
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00529 DNA chips
• CPC Class Codes: B01J 2219/00531 essentially square
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/0059 Sequential processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00617 by chemical means
• CPC Class Codes: B01J 2219/00626 : Covalent
• CPC Class Codes: B01J 2219/00648 : by the use of solid beads
• CPC Class Codes: B01J 2219/00659 : Two-dimensional arrays
• CPC Class Codes: B01J 2219/00689 : using computers
• CPC Class Codes: B01J 2219/00695 : Synthesis control routines, e.g. using computer programs
• CPC Class Codes: B01J 2219/00711 : Light-directed synthesis
• CPC Class Codes: B01J 2219/00722 : Nucleotides
• CPC Class Codes: B01J 2219/00725 : Peptides
• CPC Class Codes: B01L 7/52 : with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: B82Y 10/00 : Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
• CPC Class Codes: B82Y 30/00 : Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C07B 2200/11 : Compounds covalently bound to a solid support
• CPC Class Codes: C07C 229/14 : to carbon atoms of carbon skeletons containing rings
• CPC Class Codes: C07C 229/16 : to carbon atoms of hydrocarbon radicals substituted by amino or carboxyl groups, e.g. ethylenediamine-tetra-acetic acid, iminodiacetic acids
• CPC Class Codes: C07D 263/44 : Two oxygen atoms
• CPC Class Codes: C07D 317/62 : with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to atoms of the carbocyclic ring
• CPC Class Codes: C07H 19/04 : Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
• CPC Class Codes: C07H 19/10 : with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/00 : Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07K 1/042 : characterised by the nature of the carrier
• CPC Class Codes: C07K 1/045 : using devices to improve synthesis, e.g. reactors, special vessels
• CPC Class Codes: C07K 1/047 : Simultaneous synthesis of different peptide species; Peptide libraries
• CPC Class Codes: C07K 17/06 : attached to the carrier via a bridging agent
• CPC Class Codes: C07K 17/14 : Peptides being immobilised on, or in, an inorganic carrier
• CPC Class Codes: C07K 7/06 : having 5 to 11 amino acids
• CPC Class Codes: C12Q 1/6809 : Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6816 : characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6837 : using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6874 : involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 40/10 : Libraries containing peptides or polypeptides, or derivatives thereof
• CPC Class Codes: C40B 60/14 : for creating libraries
• CPC Class Codes: G01N 15/1433 : using image recognition
• CPC Class Codes: G01N 21/6428 : Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6452 : Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G03F 7/00 : Photomechanical, e.g. photolithographic, production of textured or patterned surfaces, e.g. printing surfaces; Materials therefor, e.g. comprising photoresists; Apparatus specially adapted therefor using photoresist structures for special production processes, see the relevant places, e.g. B44C, H01L, e.g. H01L21/00, H05K
• CPC Class Codes: G03F 7/26 : Processing photosensitive materials; Apparatus therefor G03F7/12 - G03F7/24 take precedence
• CPC Class Codes: G03F 7/265 : Selective reaction with inorganic or organometallic reagents after image-wise exposure, e.g. silylation
• CPC Class Codes: G03F 7/38 : Treatment before imagewise removal, e.g. prebaking G03F7/265 takes precedence
• CPC Class Codes: G11C 13/0014 : comprising cells based on organic memory material
• CPC Class Codes: G11C 13/0019 : comprising bio-molecules
• CPC Class Codes: Y02P 20/55 : Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
• CPC Class Codes: Y10S 436/809 : Multifield plates or multicontainer arrays

Abstract

A method for synthesizing oligonucleotides on a solid substrate. The method provides for the irradiation of a first predefined region of the substrate without irradiation of a first predefined region of the substrate. The irradiation of a second predefined region of the substrate. The irradiation step removes a protecting group therefrom. The substrate is contacted with a first nucleotide to couple the nucleotide to the substrate in the first predefined region. By repeating these steps, an array of diverse oligonucleotides is formed on the substrate.

Claims

1. A method for synthesizing oligonucleotides on a solid phase comprising the steps of:

a) providing a substrate as the solid phase, wherein said substrate comprises oligonucleotide molecules immobilized on a surface thereof, said oligonucleotide molecules coupled to a photoremovable protecting group;

b) irradiating a first predefined region of said substrate without irradiating a second predefined region of said substrate to remove said protecting group from said oligonucleotide molecules in said first region; and

c) contacting said substrate with a first nucleotide to couple said first nucleotide to said oligonucleotide molecules in said first predefined region, said first nucleotide having a nucleotide protecting group thereon, forming a first oligonucleotide on said substrate in said first predefined region that is different from an oligonucleotide in said second predefined region.

2. The method as recited in claim 1 wherein said step of irradiating is a step of masking a light source with a mask placed between said light source and said substrate, said mask comprising first transparent regions and second opaque regions, said transparent regions transmitting light from said source to at least said first predefined region, and said opaque regions blocking light from said source to at least said second predefined region.

3. The method as recited in claim 1 wherein said first and second regions each have total areas less than about 1 cm².

4. The method as recited in claim 1 wherein said steps of irradiating are conducted with a monochromatic light.

5. The method as recited in claim 1 wherein said steps of irradiating and contacting are repeated so as to synthesize 10³ different oligonucleotides on said substrate.

6. The method as recited in claim 1 wherein the step of irradiating a first predefined region is a step of irradiating half of a region of said substrate irradiated in a prior synthesis step, and not irradiating half of said region irradiated in a prior synthesis step.

7. The method as recited in claim 1 wherein said steps a) and b) are repeated to synthesize more than 1,000 different oligonucleotides on different synthesis regions of said substrate, each of said different oligonucleotides occupying an area of less than about 10^-2 cm².

8. A method of synthesizing oligonucleotides comprising the sequential steps of:

a) generating a pattern of light and dark areas by selectively irradiating at least a first area of a surface of a substrate, said surface comprising immobilized nucleotides on said surface, said nucleotides coupled to a photoremovable protective group, without irradiating at least a second area of said surface, to remove said protective group from said nucleotides in said first area;

b) simultaneously contacting said first area and said second area of said surface with a first nucleotide to couple said first nucleotide to said immobilized nucleotides in said first area, and not in said second area, said first nucleotide having said photoremovable protective group;

c) generating another pattern of light and dark areas by selectively irradiating with light at least a part of said first area of said surface and at least a part of said second area to remove said protective group in said at least a part of said first area and said at least a part of said second area;

d) simultaneously contacting said first area and said second area of said surface with a second nucleotide to couple said second nucleotide to said immobilized nucleotides in at least a part of said first area and at least a part of said second area; and

e) performing additional irradiating and nucleotide contacting and coupling steps so that a matrix array of at least 100 different oligonucleotides is formed on said surface, each different oligonucleotide synthesized in an area of less than 0.1 cm², whereby said different oligonucleotides have sequences and locations on said surface defined by the patterns of light and dark areas formed during the irradiating steps and the nucleotides coupled in said contacting steps.

9. The method as recited in claim 8 wherein said substrate is selected from the group consisting of Langmuir Blodgett film, glass, germanium, silicon, (poly)tetrafluorethylene, polystyrene, gallium arsenide, gallium phosphide, silicon oxide, silicon nitride, and combinations thereof.

11. The method as recited in claim 8 wherein each of said different oligonucleotides is contained within an area of between about 1 um² and 10,000 um².

12. The method as recited in claim 8 wherein the irradiating step c) further comprises steps of:

i) placing a mask adjacent to said substrate, said mask having substantially transparent regions and substantially opaque regions at a wavelength of light; and

ii) illuminating said mask with a light source, said light source producing at least said wavelength of light, said mask permitting illumination of half of said substrate which was illuminated and half of said substrate which was not illuminated in said step a).

13. The method as recited in claim 8 wherein said additional steps are performed so as to synthesize 10³ different oligonucleotides in 10³ respective preselected regions on said substrate.

14. The method as recited in claim 8 wherein said additional steps are performed so as to synthesize 10⁶ different oligonucleotides in 10⁶ respective preselected regions on said substrate.

15. The method of claim 8 wherein said additional steps are performed so that at least 1,000 different oligonucleotides are synthesized on said surface, and each different oligonucleotide is contained within an area less than about 1×

10^-3 cm².

16. The method of claim 8 wherein said immobilized nucleotides are attached to said surface by a linker selected from the group consisting of aryl acetylene and ethylene glycol.

17. The method of claim 8 wherein said immobilized nucleotides are attached to said surface by polyethylene glycol.

10. The method as recited in claim 10 wherein said protective group is selected from the group consisting of 6-nitroveratryloxycarbonyl, 2-nitrobenzyloxy carbonyl, dimethyl dimethoxybenzyloxy carbonyl, 5-bromo-7-nitroindolinyl, o-hydroxyalpha-methyl cinnamoyl, 2-oxymethylene anthriquinone, and mixtures thereof.